This application relates to a method apparatus for sampling blood for use in testing for either research or for diagnostic use.
Multiple blood samples are used for clinical trials for pharmacokinetic analyses. These samples are often collected by sampling whole blood freezing and then processing the frozen blood later. Frozen blood requires a 200-250 ul sample of blood to be taken. This sample size limits the number of time-points which can be taken from a single animal due to the limited blood volume of small animals such as rats. Furthermore small volumes of blood samples are desired when dealing with critically ill patients. Moreover, there are high costs involved with the freezing transportation and processing of whole blood.
Blood samples are also collected using a bloodspot technique which requires smaller sample volumes, typically 45-60 ul for humans and 15 ul for rats, although evolving analytical techniques are using samples using 10-15 ul of human blood and smaller. Referring to FIG. 1, samples are taken from the subject usually by ‘finger pricking’ the individual and then sampling the evolved blood using a glass capillary 5. Once a desired quantity of blood is taken (45-60 ul) then the blood from the capillary 5 is carefully transferred to a ‘blood spot card’ 7 such as Watman's FDA Eulte, using 15 ul aliquots spots across four spots. Care must be taken not to contaminate the card and not to touch the card with the capillary except for the pre-designated portions where the sample is to be deposited. After blood is taken and spotted, a known concentration of an internal standard is sprayed onto the spotted card and then accurately punching disks (2-6 mm diameter) out of the blood spot or multiple blood spots. Once sampling is complete, the cards 7 are dried in air before transferring or mailing to labs for processing. Because the blood is dried, not only do some enzymatic processes cease preventing further breakdown before testing or during storage, but dried blood is not considered hazardous and no special precautions need be taken in handling or shipping. Once at the analysis site, circular discs containing the dried blood are punched out of the card and the internal standard and drug (and/or metabolite) are extracted from the disks into a supernatant which is then analyzed usually by liquid chromatography mass spectrometry.
When a card is used for direct sample collection from a wound (e.g. a neonatal heel prick or a finger prick) there is risk for collection of too much blood on the card which will lead to an overlapping of samples from the spots. Additionally, if blood flow is insufficient a non-homogenous sample can be collected (multiple small spots instead of a single large spot). This will lead to difficulty in obtaining a sub-punch from the card that is representative of the entire spot. Additionally, various chemical treatments of card materials can lead to separation of the PCV and serum during the drying process leading to non-homogenous sampling.
There are drawbacks, however, to the downstream processing of blood spots. One is in the area of sample quantitation. It is difficult to sample precise volumes using traditional glass capillaries, particularly directly from an animal or patient blood bolus. Air bubbles in capillaries can result in different capillary volumes being deposited on the cards, leading to different volumes when the card is punched. While use of micropipettes (15 ul sample) can successfully create accurate spot volumes in carefully controlled settings, in practice these have proven to be unreliable.
Another drawback with the punching technique is that it relies on a constant sample viscosity in the expectation that the sample will spread uniformly on the sample card. A constant viscosity results in blood spot diameters remaining constant when equal volume samples are administered to the cards. Unfortunately, viscosity varies significantly because of differing hematocrit (Ht or HCT) or packed cell volume (PCV) levels in the blood. Samples with high hematocrit levels form smaller diameter spots on the bloodspot papers, leading to different concentrations of blood within the fixed diameter of the spots sampled. PCV levels are believed to show around a 45% variance in spot diameters. As internal standards are sprayed onto the spotted blood this could result in a 45% error in quantitation. A further problem is that the blood is placed in marked areas on the cards, but often the person sampling the blood misses the mark and blood goes outside the marked area, making it difficult to accurately locate the circular punch over the blood spot. Even if the blood spot is centered in the card, the person punching the card may not center the punch, resulting in variable sample size. Further, the punching often shears the card and that often shakes dried blood loose, and if the punch cuts across a portion of the blood spot that also causes dried blood to be ejected into the air or work area.
Moreover, the blood spots are placed on rectangular cards which are difficult to manipulate by automated equipment, thus requiring extensive, expensive and time consuming manual handling and processing. Automated handling equipment can be acquired for the specially shaped cards, but it is custom made, expensive, and of limited application.
There is thus a need for an improved method and apparatus for use in blood sampling that reduces or eliminates one or more of the above errors and difficulties.